Supplementry Figure S Tissue weights (g).... Liver Hert Brin Pncres Len mss (g) 8 6 -% +% 8 6 Len mss Len mss (g) (% ody weight) Len mss (% ody weight) c Tiilis nterior weight (g).6...... Qudriceps weight (g)...8.6... d Femur Length (mm) 8 6-6% Femur length (mm) -% Supplementry Figure S: Tissue weights nd one length of month old control nd mle mice () Liver, hert, rin nd pncres weight from months old control nd mle mice. Results re men ± SEM of nimls/group for liver nd 6 nimls/group for hert, rin nd pncres. () Len mss mesured y DEXA scns from months old control nd mle mice. Results re men ± SEM of 6 nimls/group. (c) Skeletl muscle weights from months old control nd mle mice. Results re men ± SEM of nimls/group. (d) Femur length mesured y DEXA scns (left, n=6) or y dissection (right, n=) from months old control nd mle mice.
Supplementry Figure S.. 6 weeks old.6. PG (g).... SC (g).... BAT (g)... Liver (g).8.6... PG SC BAT Liver Supplementry Figure S: Body weight, tissue weights nd histology of 6 week-old control nd mle mice () Perigondl (PG), inguinl sucutneous (SC), rown dipose tissue nd liver weights in 6 weeks old control nd mle mice. Results re men ± SEM of 6 nimls/group. () Hemtoxylin nd eosin stined sections of dipose tissues nd liver from 6 weeks old control nd mle mice. Scle r = m. Sttisticl significnce ssessed y two-tiled Student s t test, p<. etween control nd mice.
Supplementry Figure S. IR mrna levels.... IGFR mrna levels...8.6....8.6... Tissue Adip SVF Tissue Adip SVF Brown Perigondl Sucutneous ft dipose tissue (PG) dipose tissue (SC) Yer Old mice Tissue Adip SVF Tissue Adip SVF Brown Perigondl Sucutneous ft dipose tissue (PG) dipose tissue (SC) Tissue Adip SVF Tissue Adip SVF Brown Muscle Brin Liver Perigondl Sucutneous ft dipose tissue (PG) dipose tissue (SC) Months Old mice Tissue Adip SVF Tissue Adip SVF Brown Muscle Brin Liver Perigondl Sucutneous ft dipose tissue (PG) dipose tissue (SC). Yer Old mice Months Old mice P mrna levels..8.6... Tissue Adip SVF Tissue Adip SVF Brown Perigondl Sucutneous ft dipose tissue (PG) dipose tissue (SC) Tissue Adip SVF Tissue Adip SVF Brown Muscle Brin Liver Perigondl Sucutneous ft dipose tissue (PG) dipose tissue (SC) Yer Old mice Months Old mice Supplementry Figure S: IR nd IGFR expression in control nd tissues IR, IGFR nd P mrna undnce ws mesured y rel time PCR in intct dipose tissues, isolted dipocytes nd strom vsculr frction s well s skeletl muscle (gstrocnemius), rin nd liver from months old or yer old mle control nd mice on stndrd chow diet. Results re men ± SEM of -6 nimls/group. Sttisticl significnce ssessed y two-tiled Student s t test, p<. etween control nd mice.
Supplementry Figure S Glucose (mg/dl) 7 Fed Fsted Insulin (ng/ml). Fed Fsted..8.6... c Glucose (mg/dl) 6 9 Minutes d Drk Phse VO ml/ kg LBM/hr VO ml/ kg LBM/hr Light Phse Supplementry Figure S: Glucose nd insulin levels, glucose tolernce test nd oxygen consumption in control nd mice () Fed nd fsted plsm glucose nd () Insulin levels in months old control nd mle mice. Results re men ± SEM of 7-8 nimls/group. (c) Intrperitonel glucose tolernce test in months old control nd mle mice. Results re men ± SEM of 7-8 nimls/group. (d) Oxygen consumption (VO ) ws mesured y indirect clorimetry in 6 months old control nd mle mice. Results re expressed per kg of len ody mss per hour nd re men ± SEM of nimls/group. Sttisticl significnce ssessed y twotiled Student s t test, p<. etween control nd mice.
Supplementry Figure S PG Adipokines SC -CD -CD -HFD -HFD Leptin Resistin Adiponectin Leptin Resistin Adiponectin Inflmmtion PG SC c TNF MCP F/8 TNF MCP F/8 PG Differentition SC P PPAR C/EBP C/EBP P PPAR C/EBP C/EBP Supplementry Figure S: Expression of dipokines, inflmmtion nd differentition mrkers in white dipose tissues of control nd mice () Leptin, resistin nd diponectin mrna undnce ws mesured y rel time PCR in PG nd SC WAT from control nd mle mice fed either chow or high ft diet. () TNF, MCP nd F/8 mrna undnce ws mesured y rel time PCR in PG nd SC WAT from control nd mle mice fed either chow or high ft diet. (c) P, PPAR, C/EBP nd mrna undnce ws mesured y rel time PCR in PG nd SC WAT from control nd mle mice fed either chow or high ft diet. Results re men ± SEM of 6 nimls/group. Sttisticl significnce ssessed y two-tiled Student s t test, p<. etween control nd mice.
Supplementry Figure S6 Body Weight (g) 6 Body ft (g) -CD -CD -HFD -HFD Supplementry Figure S6: Body weight nd percent ody ft in chow diet or high ft diet fed yer old control nd mle mice () Body weight ssessed in months old control nd mle mice fed chow diet or high ft diet for months. Results re men ± SEM of 6 nimls/group. () Body ft content ssessed y nucler mgnetic resonnce in months old control nd mle mice fed chow diet or high ft diet for months. Results re men ± SEM of 6 nimls/group. Sttisticl significnce ssessed y two-tiled Student s t test, p<. etween control nd mice, p<. etween CD nd HFD.
Supplementry Figure S7 C/EBP mrna WT DKO PPAR mrna WT DKO 6 8 hours 6 8 hours C/EBP mrna C/EBP mrna 6 8 6 8 Supplementry Figure S7: Expression pttern of trnscription fctors during the first hours of dipocyte differentition mrna undnce ws mesured y rel time PCR in WT nd DKO cells during the first hours of induction of the differentition. Results re men ± SEM of independent experiments. Sttisticl significnce ssessed y two-tiled Student s t test, p<. etween WT nd DKO cells.
Supplementry Figure S8 d Temperture (C) mrna (fold chnge) Temperture (C) 8 7 6 6 8..... Minutes FIRKO Supplementry Figure S8: Cold exposure in ft specific IR or IGFR KO mice nd ody temperture nd mrna undnce in BAT of control nd mice () Rectl temperture ws mesured in months old control nd mle mice mintined t room temperture (n= per group). () Rectl temperture ws mesured in months old control nd dipose specific IR KO (FIRKO) mle mice or C) in months old control nd dipose specific IGFR KO mle mice every min for hours fter exposure to C environment (n=8 per group). (c) mrna undnce ws mesured y rel time PCR in BAT from months old mle control nd mice. Results re men ± SEM of 6 nimls/group. Sttisticl significnce ssessed y two-tiled Student s t test, p<. etween control nd mice. c Temperture (C) 6 8 Minutes Dio Elovl Cox UCP UCP Adr Adr IGFRKO
Supplementry Figure S9 UCP mrna (fold chnge)...... CL6 chronic tretment (dily for weeks) Cide mrna (fold chnge) 8 6 6..... Elovl mrna (fold chnge) PG SC BAT CL6 chronic tretment Sline treted Supplementry Figure S9: Appernce of systemic rown dipocytes in white dipose tissues of control nd mice chroniclly treted with CL6 () UCP, Cide nd Elovl mrna undnce ws mesured y rel time PCR in PG, SC nd BAT in control nd mice injected dily with either sline or CL6 ( g/g ody weight/dy) intrperitonelly for weeks. Results re men±sem of nimls/group nd re expressed s fold chnge over the sline group. Sttisticl significnce ssessed y two-tiled Student s t test, p<. etween control nd mice, p<. etween sline nd CL6 treted group. () -UCP immunohistochemistry stining of SC dipose tissues from control nd mle mice injected dily with CL6 ( g/g ody weight/dy) for weeks. Scle r = m.
Supplementry Tle S Rel time PCR primers IR FW CCACCAATACGTCATTCACAAC RV GGGCAGATGTCACAGAATCAA IGFR FW AGGAGAAGCCCATGTGTGAG RV GTGTTGTCGTCCGGTGTGT Leptin FW GGG CTT CAC CCC ATT CTG A RV TGG CTA TCT GCA GCA CAT TTT G Adiponectin FW GATGGCACTCCTGGAGAGAA RV TCTCCAGGCTCTCCTTTCCT Resistin FW CTG TCC AGT CTA TCC TTG CAC AC RV CAG AAG GCA CAG CAG TCT TGA C/EBP FW CAAGAACAGCAACGAGTACCG RV GTCACTGGTCAACTCCAGCAC C/EBP FW CCAAGAAGACGGTGGACAA RV CAAGTTCCGCAGGGTGCT C/EBP FW ATCGACTTCAGCGCCTACA RV GCTTTGTGGTTGCTGTTGAA PPAR FW TCAGCTCTGTGGACCTCTCC RV ACCCTTGCATCCTTCACAAG P FW GATGCCTTTGTGGGAACCT RV CTGTCGTCTGCGGTGATTT FAS FW GAGGACACTCAAGTGGCTGA RV GTGAGGTTGCTGTCGTCTGT HSL FW ACG GAT ACC GTA GTT TGG TGC RV TCC AGA AGT GCA CAT CCA GGT ATGL FW ACTGTGGCCTCATTCCTCCT RV AACTGGATGCTGGTGTTGGT GLUT FW TGATTCTGCTGCCCTTCTGT RV GGACATTGGACGCTCTCTCT Adr FW CGTCCGTCGTCTCCTTCTAC RV CGCAGCTGTCGATCTTCTTT Adr FW GGAATTTTGGCAACTTCTGG RV ACTCGGGCCTTATTCTTGGT Adr FW GCT GAC TTG GTA GTG GGA CTC RV TAG AAG GAG ACG GAG GAG GAG TNF FW ACGTGGAACTGGCAGAAGAG RV GGCCATAGAACTGATGAGAGG MCP FW AGGTCCCTGTCATGATTCTG RV GCTGCTGGTGATCCTCTTGT F/8 FW TGGATGAGTGCTCCAGGAAT RV GATGGCCAAGGATCTGAAAA
Dio FW CAGTGTGGTGCACGTCTCCAATC RV TGAACCAAAGTTGACCACCAG PGC-α FW CCCTGCCATTGTTAAGACC RV TGCTGCTGTTCCTGTTTTC mttfa FW AGT TCC CAC GCT GGT AGT GT RV GCG CAC ATC TCG ACC C Nrf FW CAG CAA CCC TGA TGG CAC CGT GTC G RV GGC CTC TGA TGC TTG CGT CGT CTG G COX FW TCTCCCCTCTCTACGCATTCT RV TCATTGGTGCCCTATGGTTT Elovl FW GGACTTAAGGCCCTTTTTGG RV TTCCGCGTTCTCATGTAGGT PRDM6 FW ACATCCGTGTAGCGTGTTCC RV GCACCAACAGTTCCTCTCCA CIDEA FW ATCACAACTGGCCTGGTTACG RV TACTACCCGGTGTCCATTTCT UCP FW CTGCCAGGACAGTACCCAAG RV TCAGCTGTTCAAAGCACACA UCP FW CAGGTCACTGTGCCCTAACCA RV CACTACGTTCCAGGATCCCAA UCP FW GACTATGGATGCCTACAGAACC RV ACTCCAGCAACTTCTCCTTG TBP FW ACCCTTCACCAATGACTCCTATG RV TGACTGCAGCAAATCGCTTGG